Terpenes are found in most organisms (microorganisms, animals and plants). These compounds are made up of five carbon units called isoprene units and are classified by the number of these units present in their structure. Thus monoterpenes, sesquiterpenes and diterpenes are terpenes containing 10, 15 and 20 carbon atoms respectively. Diterpenes, for example, are widely found in the plant kingdom and over 2500 diterpene structures have been described (Connolly and Hill, Dictionary of terpenoids, 1991, Chapman & Hall, London). Terpene molecules and their oxidized derivatives have been of interest for thousands of years because of their flavor and fragrance properties and their cosmetic, medicinal and antimicrobial effects. Plant extracts obtained by different means such as steam distillation or solvent extraction are used as source of oxidized derivatives of terpene molecules. Alternatively, terpene molecules found in plant extracts or obtained by biosynthetic processes are oxidized using chemical and enzymatic processes.
Enzymatic oxidation of terpenes often involves enzymes called cytochrome P450s (P450s), which are typically capable of catalyzing the transformation of a hydrophobic substrate, such as a terpene molecule, in a more hydrophilic one. Cytochrome P450 enzymes form a superfamily of hemoproteins found in bacteria, archaea and eukaryotes. In one of the most common activities, cytochrome P450 acts as a monooxygenase, by inserting one oxygen atom of molecular oxygen into a substrate molecule, while the other oxygen atom is reduced to water.
This catalytic reaction requires two electrons for the activation of molecular oxygen. P450s from eukaryotes use NADPH as the external reductant and source of electrons. The two electrons are transferred one at a time to the cytochrome P450 active site and this transfer requires an electron donor protein, a cytochrome P450 reductase (CPR). One CPR is not specific for one cytochrome P450. A CPR is the electron donor protein for several P450s in a given organism. In addition, a CPR from one organism can act as the electron donor protein for P450s from other organisms. In some cases P450s can also be coupled to a cytochrome b5 protein that can act as the electron donor protein or can improve the efficiency of the electron transfer from the CPR to the P450. In eukaryotic cells and particularly in plants, P450s and CPRs are generally membrane-bound proteins and are associated with the endoplasmic reticulum. These proteins are anchored to the membrane by a N-terminal trans-membrane helix.
Many P450s have low substrate specificity and are therefore able to catalyze the oxidation of many diverse structures such as for example different terpene molecules. Most of these enzymes have a particular regio- and stereo-selectivity with a given substrate but they often produce a mixture of several products from a particular substrate. Such P450s are usually involved in the breakdown and detoxification of molecules such as xenobiotics and are generally found in bacteria and animals. On the other hand, P450s involved in biosynthetic pathways show usually a specificity for certain types of substrates and regio- and stereo-selectivity. This is the case for most plant P450s.
A large number of P450s can be found in nature and particularly in plants. One plant genome can contain several hundreds of genes encoding for P450s. Many plant P450s have been characterized but considering the extremely large number of P450s present in plants, most of their functions remain unknown.
It is therefore desirable to search for new P450s capable of catalyzing new enzymatic reactions, so as to provide enzymatic production of new oxygenated compounds or for producing oxygenated compounds through different reaction types, for example from different substrates, which may be more easily accessible.
Several P450s have already been characterized. In particular, cytochromes P450 having a certain percentage of sequence identity with the cytochrome P450 of the present invention have been reported to use terpene molecules as substrates.
The closest P450s to that of the present invention are P450s from Sorghum bicolor, among which the closest sequence shares 67% identity with the amino acid sequences described herein (Accession number EER94164).
Among the oxygenated terpenes produced by the cytochrome P450 of the present invention, some are very useful in the field of perfumery and flavoring. In particular khusimol, which is produced by hydroxylation of zizaene, is one of the key components of vetiver oil and is in itself a valuable perfuming ingredient. Oxidation of zizaene using the cytochrome P450 of the present invention provides an advantageous alternative to isolation of khusimol from vetiver oil, which is a difficult and expensive process. To the best of our knowledge, no enzymatic process for the production of khusimol is known. Several other valuable perfuming and flavouring ingredients, for which no enzymatic synthesis is known to date, can be prepared using the cytochrome P450 of the present invention as will be described below.
Other oxygenated terpenes produced by the cytochrome P450 of the present invention are useful for other purposes such as drugs or agrochemical products. The cytochrome P450 of the present invention therefore opens a new biosynthetic route to diverse molecules having interesting properties useful in various fields of the industry and being difficult or even impossible to isolate from nature and difficult or impossible to produce by organic synthesis.
It is an objective of the present invention to provide methods for making oxygenated terpenes, in particular khusimol, in an economic way. Accordingly, the present invention has the objective to produce oxygenated terpenes while having little waste, a more energy and resource efficient process and while reducing dependency on fossil fuels. It is a further objective to provide enzymes capable of oxidizing terpene molecules, such oxidized products being useful as perfumery and/or aroma ingredients.